Cellular and Molecular Biotechnology Research Institute(CMB), AIST

Phosprof
User Guide

Version 1.1
2022/07/13

Contents1

Introduction ................................................................................................... 22

Signature Protein........................................................................................... 33452.1

Preparation ............................................................................................. 3

2.2

Pathway Selection ................................................................................... 3

2.3

Visualization ........................................................................................... 42.4

Detailed information ............................................................................... 6

Pathway ......................................................................................................... 73.1

Preparation ............................................................................................. 83.2

Visualization ........................................................................................... 8Pathway comparison .................................................................................... 124.1

Preparation ........................................................................................... 124.2

Compared Pathways ............................................................................. 13

Drug Search ................................................................................................. 145.1

Input ...................................................................................................... 145.2

Search Results....................................................................................... 156Drug List ...................................................................................................... 167Contact ......................................................................................................... 1611Introduction

Phosprof (phosphorylation profiling DB) is a database that presents cellularresponses to representative drugs as significant pathways. It is based on theanalysis of collected experimental data on the phosphorylation activities using
protein arrays. Measurement of the phosphorylation activity allows direct
evaluation of the signal transduction activity and estimation of the pathways
responsible for a particular cellular event. A comparison between the various
approved drugs with known target phenotypes can be helpful for further
investigation of the drug functions and for drug development.
Phosprof：http://phosprof.medals.jp/2

2Signature Protein
Phosphorylation reactions in the protein array allow measurement of thekinase activity of the specimen. We summarize the resultant ‘phosphorylation
profiles’ of lysates of cells treated with 94 drugs by highlighting the proteinsshowing significant phosphorylation level changes induced by the drugtreatment, on the selected pathway map.

2.1 Preparation
Signature Proteins can be browsed on the top page of Phosprof by selecting thecell line tested (click button) and the drug of interest (select from a drop-downlist). The tested cell lines are MCF-7 (human breast cancer cell line) and K562
(immortalized human myelogenous leukemia cell line). The 94 tested drugs arefrom the FDA-approved Drug Library (Selleck Pfizer Licensed Library, L2400
Selleck chem).

2.2 Pathway Selection
On the next page, pathway maps from the four pathway groups are selected tovisualize significant proteins colored on the selected pathway map. For
legibility, we provide two types of pathway maps: full pathways containing all
the 1,376 proteins as pathway nodes, and simple pathways with integrated
nodes of complex proteins or protein groups that are not directly connected bymain binaries along the membrane-to-nucleotide direction. A full or simple
map according to is chosen acc as required.

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2.3 Visualization
Proteins with significant changes are shown in the pathway map. Proteins
with increased (Up) or decreased (Down) tyrosine phosphorylation levels areshown as orange and blue nodes, respectively. The proteins of the pathway arealigned in the membrane (top)-to-nucleus (bottom) direction, referring to theReactome database. The protein nodes of the pathway maps are connected oneby-one with edges, if the interactions between two proteins are known. Thedata type (Std [standardized] or Raw [raw-data]), analysis type (RP [rankproduct] or Pearson), and threshold (by rank (enter number in the blank spaceleft of ‘Top’) or P-value) can be selected using the boxes in the upper rightcorner.

4The protein of interest can be searched on the map using the box in thebottom-right corner of the screen.

A list of significant pathways that include each protein can be visualized byhovering the cursor over each node.

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2.4 Detailed information
Detailed information on the protein of interest can be visualized by doubleclicking on the node on the pathway map. The ‘Detail’ page of the selected
protein provides their gene symbol, gene ID, and amino acid sequence. The
disorder score is plotted along the amino acid sequence as the red line on thegraph titled ‘Sequential information.’ The green vertical lines in the graphcorrespond to the tyrosine residues of the protein. The tyrosine residues thatare annotated to be ‘Phospho-tyrosine’ in UNIPROT are indicated by light-bluecircles.

‘Structural Information’ provides links to the PDB site of the protein via UNIPROT.

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The PDB ID is shown if available and linked to the 3D image. The PDB ENTITY with thelongest sequence was selected. In the 3D image, tyrosine residues are illustrated as a balland-stick model.

3Pathway7

Based on the distribution of the significant proteins, a pathway analysis isperformed to estimate the significance of the 376 pathways. To systematicallyreview the significant pathways in the interconnected pathway network, theyare highlighted in the hierarchical tree in the ‘Pathway’ section. The pathway
hierarchy is constructed according to the Reactome database, and every
pathway node is linked to the original Reactome pathway site that provides
more detailed information.

3.1 Preparation
On the ‘Pathway’ section, select the tested cell line and drug of interest, andthen click ‘SUBMIT’.

3.2 Visualization
Pathways are displayed in a tree form, and each pathway node is connected tothe Reactome database, which provides more detailed information. Significant
pathways with up- or downregulated signature proteins are shown as orangeor blue-colored nodes, respectively. The data type, analysis type, thresholds ofsignature proteins (number of signature proteins), and pathway analysis (pvalue) can also be selected using the boxes in the upper right corner.

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The pathways are shown in a tree form as they form a hierarchical network.
The top stratum pathway is signal transduction, since all the 376 pathways
examined in Phosprof belong to this pathway group. Significant pathways areshown as colored boxes, and the pathways of the higher stratum are also
shown in the same colors, but in their paler shades.

9On clicking the ‘Up’ button, significant pathways with upregulated signature
proteins are visualized as orange boxes.

On clicking the ‘Down’ button, significant pathways with downregulated
signature proteins are visualized as blue boxes.

10On clicking the UpDown button, significant pathways with both up- anddownregulated signature proteins are visualized as orange and blue boxes,respectively.

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4Pathway comparison
To examine the similarities and differences in functions among drugs, thesignificant pathways for each drug are compared and displayed in a Venn’s
diagram in the Compare Pathway section. The Compare Pathway tool enables
comparison of significant pathways among five or fewer drugs of interest, withvariable thresholds. The numbers on the Venn’s diagram can be clicked on tovisualize the pathway list.

4.1 Preparation
A drug can be selected to compare it with the drugs on the drug list on the left
by checking the boxes.

124.2 Compared Pathways

Common and uncommon pathways among the selected drugs are shown in a
Venn’s diagram. As the pathways form a structural hierarchy network, they
can be compared at various depths of the pathway stratum by selecting apathway stratum of 1–5 to search for common features. All the analyzed
proteins belonged to signal transduction, which is the top stratum (pathwaystratum = 1). On clicking on an area of the Venn’s diagram, the corresponding
pathway list is shown on the right.

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5Drug Search
The phosphorylation profile data of compounds not analyzed in Phosprof canalso be searched for based on similarity with the drugs that were analyzed. On
entering the SMILES or SDF of the drug of interest in the Drug Search section,
the similarity with drugs in Phosprof is represented as the Tanimoto coefficientscore.

5.1 Input
The tested cell line can be selected, and the information of the drug ofinterest can be used as input in a SMILES or SDF form followed by clicking
on ‘Search.’14

5.2 Search Results
When the input forms are accepted and the calculations are completed, 94
drugs are aligned in the list based on their Tanimoto coefficient scores. Drugs
are linked with the signature section to check the properties of the drug in
Phosprof that share analogous features with the drug of interest used as input.

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6Drug List
All the 94 drugs examined in Phosprof are listed in the drug list section withdetailed information, including the formulas, CAS numbers, and SMILES, withlinks to PubChem.

7Contact

Phosprof
URL: https://phosprof.medals.jp
If you have any questions or comments, please contact the following e-mailaddress.
mail: h-kagiwada (+@aist.go.jp)
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Acknowledgments:
・This work was supported by the Japan Agency for Medical Research andDevelopment (Grant Nos. 21ae0101074s0203).
・This research was supported in part by the Life-Science Database Integration
Project at the Japan Science and Technology Agency (JST).
Cellular and Molecular Biotechnology Research Institute (CMBRI),
National Institute of Advanced Industrial Science and Technology (AIST),Tokyo Waterfront Bio-IT Research Building
2-4-7 Aomi, Koto-ku,Tokyo,135-0064, Japan

Copyright ©CMBRI, AIST, 2022 All Rights Reserved.

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